A pan-coronavirus fusion inhibitor targeting the HR1 domain of human coronavirus spike

Xia, Shuai; Yan, Lei; Xu, Wei; Agrawal, Anurodh Shankar; Algaissi, Abdullah; Tseng, Chien-Te K.; Wang, Qian; Du, Lanying; Tan, Wenjie; Wilson, Ian A.; Jiang, Shibo; Yang, Bei; Lu, Lu

To systematically assess the inhibitory activities of these peptides against different HCoVs, we developed multiple cell-cell fusion assays that were mediated by the S protein of various HCoVs (fig. S1D). Consistent with previous results, MERS-HR2P had inhibitory activity against cell-cell fusion mediated by MERS-CoV S protein with a concentration for 50% inhibition (IC 50 ) of 1.01 M, whereas it showed little inhibitory activity in other HCoV S-mediated cellcell fusion assays even with concentrations up to 5 M ( Fig. 1D and table S1). Similarly, SARS-HR2P specifically inhibited SARS-CoV S-mediated cell-cell fusion with an IC 50 of 0.52 M. On the other hand, HR2P peptides derived from the two -HCoVs, i.e., 229E and NL63, showed potent and broad inhibitory activity against -HCoV S-mediated cell-cell fusion with IC 50 values ranging from 0.13 to 0.51 M or from 0.21 to 0.56 M, respectively, but no effective inhibitory activity against -HCoVs (including MERS-CoV, SARS-CoV, and OC43) S-mediated fusion ( Fig. 1D and table S1 ). OC43-HR2P exhibited broad and potent fusion inhibitory activity with IC 50 values of 0.39, 0.54, and 0.66 M against MERS-CoV, SARS-CoV, and OC43, respectively ( Fig. 1D and table S1 ). Unexpectedly, OC43-HR2P, a -HCoV HR2-derived peptide, and thus 14 residues shorter than 229E-HR2P and NL63-HR2P, exhibited effective activity against -HCoVs with an IC 50 of 0.84 M on 229E-S-mediated cell-cell fusion and an IC 50 of 0.94 M on NL63-S-mediated cell-cell fusion. Among all HR1Ps, only 229E-HR1P exhibited moderate inhibitory effects on 229E-S-and NL63-S-mediated cell-cell fusion (Fig. 1D) . Thus, compared to HR1Ps, all HCoV HR2-derived peptides exhibited excellent self-specific fusion inhibitory activity, whereas OC43-HR2P showed broad-spectrum and potent fusion inhibitory activity against both -HCoV and -HCoV S-mediated cell-cell fusion ( Fig. 1D and table S1 ). We also measured the -helicity of OC43-HR2P and HCoV-HR1Ps by circular dichroism and found that the peptides alone exhibited limited -helicity ranging from 11.3 to 42.3% ( fig. S1E ). In contrast, the mixtures of OC43-HR2P and each of these HR1 peptides, respectively, exhibited high -helicity (70.9 to 86.7%) with melting transition temperature (T m ) values that ranged from 48.5° to 91.5°C ( fig. S1E ), further implying that OC43-HR2P can bind HR1s of different HCoVs and form stable complexes, thereby blocking S protein-mediated fusion.